APOBEC3 (A3) proteins are members of an innate immune response that provide a defense against HIV-1 and other pathogens. In the absence of the HIV-1 protein Vif (Vif1), the A3 proteins are incorporated into virions in the virus producer cells and inhibit viral replication by deaminating cytidines in the minus-strand of viral DNA during reverse transcription in the target cells, resulting in extensive G-to-A hypermutation of the viral genome. In addition to inactivating most of the viral genomes through lethal hypermutation, we and others have shown that A3G and A3F also inhibit viral DNA synthesis and integration. To overcome these host defenses, Vif1 binds to the A3 proteins and targets them for proteasomal degradation, preventing their incorporation into virions. Defining the interactions of Vif1 with A3G and A3F at the molecular level could provide two potential targets for the development of antiviral drugs to suppress A3G and A3F degradation. ____Our goal is to understand the structure and function of Vif and A3 proteins. We will gain insights into the structures of Vif:A3 complexes through mutational and comparative analyses and generate reagents for structural studies. We recently determined that Vif1 and HIV-2 Vif (Vif2) interact with A3 proteins by using completely different determinants. We determined the relative restriction potential of A3G and A3F in primary CD4+ T cells by using Vif1 mutants that specifically failed to induce degradation of A3G or A3F, and showed that A3G exerts a greater restriction effect on HIV-1 than the combined activity of A3F and A3D. In collaboration with Dr. Kei Sato (Institute for Virus Research, Kyoto University), we also determined the replication potential in humanized mice of Vif1 mutants that are deficient in inducing degradation of A3G or A3F and found that both A3 proteins potently inhibited HIV-1 replication. A3G is localized to cytoplasmic RNA processing bodies (P bodies); we previously found that Mov10, a putative RNA helicase, is also localized to P bodies and inhibits HIV-1 replication. We therefore explored the significance of A3G and Mov10 localization to P bodies to their antiviral activity and concluded that virion incorporation and antiviral activities of A3G and Mov10 do not require localization to P bodies. In collaboration with Dr. Jeffrey Lifson, we found that replication of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus, is severely restricted in pigtailed macaques; in agreement with our in vitro studies, XMRV restriction was associated with extensive G-to-A hypermutation, suggesting powerful restriction by A3 proteins. _____To identify host factors that facilitate Vif1-mediated degradation of A3G, we performed a genome-wide siRNA screen in collaboration with NIH Chemical Genomics Center (now NCATS). These studies revealed that UBA52, a fusion protein of ubiquitin and ribosomal protein L40, is a major source of ubiquitin for Vif1-mediated degradation of A3G and that a UBA52 mutant is a dominant inhibitor of Vif1-induced A3G degradation. The UBA52 mutant did not inhibit Vif2-induced degradation of A3G, indicating that Vif2 uses a different mechanism. We will define the mechanism that Vif2 uses to induce A3G degradation, and in collaboration with Dr. Eric Barklis (Oregon Health & Science University), we will use E. coli biotin ligase and mass spectrometry to identify and characterize Vif2- and Vif1-interacting proteins to gain insights into their different mechanisms. We will also characterize Vif proteins of HIV-1 groups M, N, O, and P to compare their mechanisms and interactions with human A3 proteins. The ultimate goal of these comparative studies is to generate stable Vif:A3 complexes and to determine their structures in collaboration with Dr. Yong Xiong (Yale University). _____We are studying the mechanisms by which A3 proteins inhibit viral replication and how they potentially affect viral genetic variation and evolution. Although A3G, A3F, A3D, and A3H have been shown to inhibit HIV-1 replication, it is not known whether they can copackage into the same virions and comutate the HIV-1 genomes. We found that A3G and other A3 proteins can copackage and comutate the same viral genomes and do not synergize or antagonize each other's antiviral activities. We sought to determine the potential contribution of A3-induced G-to-A hypermutation to viral genetic variation. We examined the potential for hypermutation to affect recombination frequency and induce sublethal mutagenesis in vivo, and for recombination to assort G-to-A hypermutations. Our results indicate that the contribution of A3-induced hypermutation to viral genetic variation is far less than that of error-prone viral replication. _____We have developed lentiviral vectors that can efficiently deliver Vif1-resistant A3 genes to T cells. To elucidate the evolutionary potential of Vif1 to evolve and overcome host restriction proteins, we will express Vif1-resistant A3 proteins in T-cell lines and determine the potential for Vif1 to evolve and acquire the ability to degrade these restriction factors in the presence and absence of wild-type A3 proteins. These studies will provide insights into the evolutionary interplay between HIV-1 and host restriction factors. _____In our recent collaborative study with the laboratories of Drs. Hiroshi Matsuo (Leidos Biomedical Research, Inc.) and Celia Schiffer (University of Massachusetts Medical School), the crystal structure of the catalytic domain of HIV-1 A3G in complex with single-stranded DNA (ssDNA) was determined at 1.86-angstrom resolution. To overcome weak DNA-binding affinity between A3G and the C-terminal domain (CTD), we generated a catalytically active variant of A3G-CTD that binds ssDNA stronger than wild-type. This A3G-CTD variant was co-crystallized with a 9-nucleotide ssDNA containing a 5'-TCCCA target sequence with all 9 nucleotides well resolved in the structure. The nucleotides within the 5'-TCCCA target sequence show numerous interactions with A3G-CTD, explaining the nucleotide specificity preferences. Furthermore, the backbone architecture of the protein changed upon ssDNA binding, enabling the target sequence to fit. These results provide fundamental insights into the mechanisms by which APOBEC3s recognize their specific substrate sequences. _____ In collaboration with Dr. Yong Xiong, we recently presented a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1), which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. The crystal structure of a non-preferred adenine in the -1 nucleotide-binding pocket of A3G was determined, revealing a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.